Colonization control of lactose-fermenting Salmonella typhimurium in young broiler chickens by use of dietary lactose

Inclusion of lactose in the diets of chickens has been determined to reduce cecal colonization with Salmonella typhimurium. We hypothesized, therefore, that dietary lactose may be a practical means for reducing the prevalence of Salmonella contamination of chicken products. Because some strains of Salmonella are atypical and ferment lactose, we investigated the effects of dietary lactose on cecal colonization with lactose-fermenting S typhimurium. Broiler chicks were inoculated intracloacally with Lac+ S typhimurium selected for resistance to novobiocin and rifampicin. The chicks also were inoculated orally with certain anaerobes that do not effectively inhibit colonization by S typhimurium, but do appear essential for lactose mediated inhibition of cecal colonization. Control chicks were not given dietary lactose, and chicks in the experimental group were fed a diet containing 7% lactose. Enumeration of Lac+ S typhimurium in cecal contents revealed dietary lactose to be effective at controlling this organism. Control was correlated with changes in cecal pH and increases in undissociated volatile fatty acids, especially propionic acid.     

Intracloacal Salmonella typhimurium infection of broiler chickens: reduction of colonization with anaerobic organisms and dietary lactose

The combined effect of treatments with dietary lactose plus anaerobic organisms on cecal colonization of broiler chicks by Salmonella typhimurium was evaluated. Chickens treated with a combination of anaerobic organisms and 7% dietary lactose were resistant to cecal colonization by S. typhimurium. The number of recoverable S. typhimurium cells per gram of cecal contents taken on days 10 and 15 after infection was significantly reduced. Treatment with anaerobes without the addition of lactose did not effectively control cecal colonization. Intracloacal inoculations with bacterial concentrations that varied by 10,000-fold resulted in roughly similar levels of colonization. The treatments resulted in reduced cecal pH and elevated levels of undissociated volatile fatty acids. Statistically significant correlations (P less than 0.01) were observed between the S. typhimurium concentrations in cecal material and the concentrations of undissociated fatty acids (r = -0.79, and between the bacterial counts and pH (r = 0.72).     

Effect of dietary lactose on Salmonella colonization of market-age broiler chickens

The effect of providing lactose in feed and inoculation with volatile fatty acid-producing anaerobic cultures (AC) of cecal flora on Salmonella typhimurium colonization was evaluated in broilers. One-day-old chicks were divided into four groups and provided 1) no lactose, no AC; 2) AC, no lactose; 3) AC and lactose on days 1-10; or 4) AC and lactose on days 1-40. All groups were challenged per os with 10(6) Salmonella on day 3 and with 10(8) Salmonella on day 33. Salmonella growth in the cecal contents was significantly decreased (P less than 0.01) on day 10 in the chicks provided lactose from day 1-10. However, after the removal of lactose from the diet, the chicks were susceptible to Salmonella colonization. The number of Salmonella in the ceca was significantly reduced (P less than 0.05) in the chicks provided lactose throughout the 40-day growing period. Dietary lactose decreased the pH of the cecal contents and was accompanied by marked increases in the concentrations of undissociated bacteriostatic volatile fatty acids in the cecal contents.     

Effect of dietary lactose on cecal pH, bacteriostatic volatile fatty acids, and Salmonella typhimurium colonization of broiler chicks

One-day-old broiler chicks were inoculated with volatile fatty acid producing cecal flora from adult chickens. The chicks were divided into four groups and provided 1) no lactose, 2) 2.5% lactose in water, 3) 5% lactose in feed, or 4) 10% lactose in feed, until 10 days of age. All groups were challenged at 3 days of age with 10(6) or 10(8) S. typhimurium. At 10 days, the number of Salmonella in the ceca of the chicks challenged with 10(6) Salmonella was significantly decreased (P less than 0.01) in the groups provided lactose as compared with the controls. A significant decrease (P less than 0.01) in Salmonella numbers occurred in the chicks challenged with 10(8) Salmonella and provided 10% lactose. Providing 2.5% lactose or 5% lactose failed to inhibit Salmonella growth in chicks challenged with 10(8) Salmonella. The pH of the ceca of the groups provided lactose decreased significantly (P less than 0.05) and was accompanied by significant increases (P less than 0.01) in the concentrations of bacteriostatic acetic and propionic acids. Results showed that providing dietary lactose to broiler chicks and inoculation with normal cecal flora decreased cecal pH, increased the concentrations of bacteriostatic volatile fatty acids, and inhibited Salmonella colonization.     

Effect of anaerobic cecal microflora and dietary lactose on colonization resistance of layer chicks to invasive Salmonella enteritidis.

The effect of oral inoculation with anaerobic cultures of cecal microflora and providing lactose in the feed on colonization resistance to invasive Salmonella enteritidis was evaluated in newly hatched leghorn chicks. Salmonella colonization of the ceca, tissue invasion and organ colonization, horizontal transmission, and seroconversion were significantly decreased (P less than 0.01) in chicks inoculated with cecal flora. The addition of lactose to the feed, in the absence of cecal microflora, failed to provide protection. Dietary lactose enhanced colonization resistance in chicks that were inoculated with anaerobic cultures of cecal flora. The results indicated that establishment of normal cecal flora in layer chicks together with the addition of lactose to the diet markedly increases resistance to cecal colonization and organ invasion, and decreases horizontal transmission of S. enteritidis.     

Factors influencing enhanced Salmonella typhimurium infection in Eimeria tenella-infected chickens

To investigate a possible mechanism involved in the enhancement of Salmonella typhimurium infection in chickens concurrently infected with Eimeria tenella, S typhimurium was given orally to chickens 7 days after E tenella inoculation. The number of viable S typhimurium decreased in the ceca of chickens not inoculated with E tenella, whereas the number gradually increased in the ceca of chickens inoculated with E tenella. Cecal contents were analyzed for pH value, oxidation-reduction potential, and amounts of short-chain fatty acids and bile acids. In the ceca of E tenella-inoculated chickens, the oxidation-reduction potential significantly (P less than 0.05) shifted to the oxidative phase, and the concentration of volatile fatty acids (acetic acid, propionic acid, and butyric acid) significantly (P less than 0.05) decreased. In both aerobic and anaerobic incubations, the number of viable S typhimurium in vitro decreased as the molar concentration of fatty acids increased. Experimental evidence indicated that multiplication of S typhimurium in the ceca of E tenella-inoculated chickens was associated with decreased concentrations of volatile fatty acids.     

Effect of anticoccidial and antimicrobial feed additives on prevention of Salmonella colonization of chicks treated with anaerobic cultures of chicken feces

Chicks were treated orally on the day of hatch with either fresh or frozen competitive exclusion (CE) cultures (native gut microflora). Chicks were fed either unmedicated feed or one of five commercial broiler starter rations or nine experimental feed mixtures containing varying amounts and combinations of anticoccidial and antimicrobial medicaments. After 2 days, they were challenged with approximately 10(6) colony-forming units of a nalidixic-acid-resistant strain of Salmonella typhimurium. Six days later, chicks were sacrificed and ceca were analyzed for S. typhimurium. Colonization of 2-day-old chicks was prevented or at least greatly reduced in most instances by treatment of chicks with a CE culture, but the efficacy of CE broth cultures stored at -70 C diminished over time. Not all CE cultures tested gave equal protection against Salmonella colonization, and CE cultures were more susceptible to some feed additives than others. Of the commercial or experimental feed tested, only the feed containing the combination of nicarbazin and bacitracin interfered with the protective effect of the CE culture.     

Adhesion of bacteria to the cecal mucosal surface of conventional and germ-free chickens infected with Eimeria tenella.

When Salmonella typhimurium and Clostridium perfringens were tested in conventional chickens, larger numbers of S typhimurium and C perfringens adhered to Eimeria tenella-infected ceca than to uninfected ceca. In germ-free chickens, S typhimurium and C perfringens adhered to the E tenella-infected cecal mucosa more than to the uninfected cecal mucosa, but fewer Bacteroides vulgatus and Bifidobacterium thermophilum adhered to the E tenella-infected ceca than to the uninfected ceca. Many bacteria adhered to the lesions caused by E tenella as observed by scanning electron microscopy. On the basis of our findings, we suggest that infection with E tenella upsets the balance of competitive adherence of bacteria, allowing more colonization of S typhimurium and C perfringens.     

Colonization characteristics of Campylobacter jejuni in chick ceca

We report our findings on several parameters influencing cecal colonization of chickens by Campylobacter jejuni. Thirty-five colony-forming units (CFU) of a composite culture of C. jejuni colonized the ceca of one-half of the newly hatched chicks challenged by oral gavage. A challenge dose of 3500 CFU/chick consistently colonized the ceca of all chicks challenged. Challenge doses of approximately 10(5) CFU of C. jejuni per chick resulted in consistent cecal colonization, regardless of whether the birds were challenged 1, 2, or 3 days post-hatch. Four isolates showed consistently strong cecal colonization abilities, whereas two isolates colonized the ceca in only 20 of 122 chicks when given levels of 10(5) CFU per chick. One of these poorly colonizing isolates was repeatedly transferred by fecal-oral passage through chicks; subsequently, this isolate was able to consistently colonize chicks. Competitive exclusion (CE) microflora did not diminish the colonization rates for C. jejuni. Birds treated with five different CE cultures were colonized at a rate of 81 of 84 chicks; control chicks were similarly consistently colonized (45 of 46 chicks).     

In vitro attachment of Salmonella typhimurium to chick ceca exposed to selected carbohydrates

This investigation was designed to study the effect of selected carbohydrates on the in vitro attachment of Salmonella typhimurium to the ceca of chickens 1 and 2 weeks of age. Ceca were surgically removed from chickens immediately after euthanasia, inverted on glass rods, and then rinsed with sterile saline before being exposed to S. typhimurium in a solution of saline containing the carbohydrate to be evaluated. Attachment of S. typhimurium to ceca was reduced in 1-week-old chicks in the presence of N-acetyl-D-galactosamine, L-fucose, D-galactose, L+arabinose, and D+mannose. Minimal or no reduction in attachment of S. typhimurium was noted when ceca from 2-week-old chicks were exposed to the same compounds. Carbohydrates investigated and found to be ineffective for reduction of S. typhimurium attachment to chick ceca were D+fucose and N-acetyl-D-glucosamine.     

Effect of feeding selected carbohydrates on the in vivo attachment of Salmonella typhimurium in chick ceca.

Two-day-old chicks were orally inoculated with 1 ml of Salmonella typhimurium (10(5) colony-forming units/ml) and divided into four groups. Three groups were fed 2.5% carbohydrates starting on day 1 (arabinose, galactose, and lactose), while the fourth group served as the control. Ceca were obtained from each group at 7, 14, and 21 days. At the end of 14 days, all three carbohydrates statistically reduced Salmonella recovery. However, lactose failed to reduce recovery between day 14 and day 21. Arabinose and galactose continued to show significant reductions of recovery through 21 days. No statistical difference was found between Salmonella recovery from whole ceca (with cecal material) and inverted ceca (washed free of cecal material).     

The influence of physical and environmental variables on the in vitro attachment of Salmonella typhimurium to the ceca of chickens

This investigation was designed to study the effect of exposure time, pH, age of bird, and native intestinal microflora on the in vitro attachment of Salmonella typhimurium to the ceca of chickens. Ceca were surgically removed from chickens immediately after euthanasia, and the interiors were exposed to S. typhimurium as intact ceca with contents, intact ceca rinsed free of contents, or inverted rinsed ceca. Attachment of S. typhimurium was slightly higher in washed than in unwashed ceca. Neither pH nor age of chicks affected attachment of the organism to ceca. There was no difference in attachment of salmonellae to inverted washed ceca after 1, 2, 3, 5, and 10 min exposure, but a one log difference was noted between 10 min and 30 sec.     

Effect of administration of Lactobacillus crispatus,Clostridium lactatifermentans and dietary lactose on the development of the normal microflora and volatile fatty acids in the caeca of broiler chicks

1. Lactobacillus crispatus and Clostridium lactatifermentans, both isolated from the caeca of chickens, grown together in an in vitro model system are able to ferment lactose to acetate and propionate. In this study, the capabilities of these organisms were studied in vivo. 2. The effect on concentrations of volatile fatty acids and lactate, together with the development of some bacterial groups in the caeca of chicks, was studied after oral inoculation with L. crispatus and C. lactatifermentans together with dietary lactose. For this purpose, chicks were divided into 4 groups: (i) control group, (ii) dietary lactose, (iii) L. crispatus and C. lactatifermentans, and (iv) dietary lactose together with L. crispitaus and C. lactatifermentans. 3. In general, concentrations of (undissociated) volatile fatty acids in the caeca were not significantly different in broilers receiving both bacteria and dietary lactose compared with control broilers. Concentrations of lactate in the caeca of 14-d-old broilers treated with any of the three treatments were significantly higher than in the caeca of control broilers. 4. This indicates that L. crispatus or other lactate-producing organisms were responsible for the elevated concentrations of lactic acid. Clostridium lactatifermentans has probably not colonised the caeca sufficiently to ferment this lactate further to acetate and propionate. 5. Numbers of Enterobacteriaceae and enterococci in the caeca of broilers receiving both bacteria and dietary lactose were not different from control broilers. 6. We conclude from these results that under the conditions applied in this study a mixture of L. crispatus and C. lactatifermentans with dietary lactose was able to increase lactate concentrations but was unable to increase concentrates of acetate and propionate in the caeca of broiler chicks.     

Interaction of aflatoxin with Eimeria tenella infection and monensin in young broiler chickens.

Young broiler chicks inoculated with Eimeria tenella and given a diet containing 2.5 mug aflatoxin/g had significantly higher mortality than birds with aflatoxicosis or coccidiosis alone or uninoculated controls. This effect was seen even when a light coccidial infection alone did not increase mortality or cause weight depression. In addition this higher mortality with the combination began earleir and occurred at a higher rate than did mortality from aflatoxin or cecal coccidiosis alone. Dietary monensin sodium (99 umg/g) did not completely prevent mortality and weight depression when aflatoxin and E. tenella were in combination. Aflatoxin and E. tenella singly significantly depressed three-week body weights; however, the depression was most severe when the two were in combination. Both dietary aflatoxin and E. tenella significantly reduced hemoglobin, packed cell volume, and plasma pigmentation, and in combination resulted in more severely reduced hemoglobin, packed cell volume, and plasma pigmentation. Coccidial lesion scores were significantly less for the combination of E. tenella and aflatoxicosis than for coccidiosis alone. This atypical response of the ceca to E. tenella in the presence of dietary aflatoxin was characterized by less distended ceca, very little coagulated blood in the ceca, and apparently more profuse cecal hemorrhage.     

Gamma/delta T cell response of chickens after oral administration of attenuated and non-attenuated Salmonella typhimurium strains

Poultry represents an important source of Salmonella infection in man. Despite intensive research on immunity, little is known about the involvement of T cell sub-populations in the immunological response of chickens against infection with non-host-adapted Salmonella (S.) serovars. In this study, the T cell composition of blood lymphocytes (CD4(+)CD8(+); CD4(+)CD8(-); CD4(-)CD8(+); CD8(+)TcR1(+); CD8(-)TcR1(+), CD8(+)TcR1(-)) after oral administration of the non-attenuated S. typhimurium wild-type strain 421 (infection) or the attenuated vaccine strain Salmonella vac((R)) T (immunization) to day-old chicks was investigated and compared with non-treated chickens by flow cytofluorometry. Additionally, the occurrence of T cell sub-populations (CD4(+); CD8(+); TcR1(+)(gammadelta); TcR2(+)(alphabeta(1))) in ceca, spleen and bursa of Fabricius of the birds was studied immunohistologically. Blood samples and tissues were examined between days 1 and 12 of age.Chicks inoculated with S. typhimurium 421 or Salmonella vac((R)) T showed significantly elevated percentages of CD8(+)TcR1(+) in blood on days 7, 8 and 9, or on day 8 in comparison to control animals. The CD4 to CD8 cell ratio was about 3:1 in infected animals on day 5 of age. In the organs of treated chicks the numbers of CD8(+)(gammadelta) and TcR1(+)(gammadelta) cells had markedly increased on days 4 and 5 in ceca, 8 and 9 in the bursa and 9 and 12 in the spleen. Moreover, infected or vaccinated birds revealed larger quantities of CD4(+) and TcR2(+) T cells in ceca on days 4 and 5. As shown by double staining, the TcR1(+) cells in the organs of infected animals additionally carried the CD8 antigen.In conclusion, immunization of day-old chicks with the attenuated Salmonella live vaccine strain resulted in the same changes in T cell composition as seen after infection with the non-attenuated Salmonella wild-type strain, but at a lower level. The remarkable increase of CD8(+)TcR1(+)(gammadelta) double positive cells in treated birds indicates an important role of this cell sub-population in the immunological defense of chickens against Salmonella exposure.     

Effect of carbohydrates on Salmonella typhimurium colonization in broiler chickens

The effect of carbohydrates in the drinking water of broiler chickens on Salmonella typhimurium colonization was evaluated. Results indicate that mannose and lactose (2.5%) significantly (P less than 0.05) reduced intestinal colonization of S. typhimurium by at least one-half, as compared with dextrose, maltose, and sucrose. Lactose and mannose also significantly reduced (P less than 0.01) the mean log10 number of S. typhimurium in the cecal contents. Although mannose was the most effective sugar at blocking colonization, lactose may be more practical because it is effective and costs much less than mannose. Provision of carbohydrates in the drinking water had no significant effect on weight gain.     

Reduction of Salmonella typhimurium concentration in broiler chickens by milk or whey

Whey (5%) in the feed of chicks for the first 10 days of life reduced the mean log10 number of viable S. typhimurium from 5.68 in control chickens to 3.38 in whey-fed chickens. Lactose in drinking water or reconstituted dry milk (5% wt: vol) in drinking water reduced the mean log10 number of S. typhimurium to 2.60 and 2.11, respectively. Milk (5% wt: wt) in feed was not effective in reducing S. typhimurium colonization. The lack of effect of milk in the feed is believed to be because not enough lactose was provided at the 5% (wt: wt) concentration. Lactose in whey or nonfat dried milk offers alternatives to the use of pure lactose in preventing or lowering S. typhimurium numbers in young broiler chickens.     

Effect of used litter from floor pens of adult broilers on Salmonella colonization of broiler chicks.

The effect of used pine-shaving litter from broiler floor pens on Salmonella colonization resistance was evaluated in broiler chicks. One-day-old chicks were placed in floor pens on fresh unused litter or on used litter. All chicks were challenged orally with 10(4) S. typhimurium at 3 days of age. The study was replicated in three trials with used litter that was collected and stored for 1 day (Trial 1), 4 days (Trial 2), or 50 days (Trial 3) before the start of each trial. Cecal concentrations of volatile fatty acids (VFAs) were significantly higher (P < 0.05) in chicks placed on used litter than in chicks on new litter. In all three trials, the number of Salmonella in the cecal contents and the number of Salmonella cecal-culture-positive chicks was significantly lower (P < 0.01) at 10 days and 20 days of age in the chicks on used litter than in the chicks on new litter. The results indicated that newly hatched chicks reared on used litter had higher cecal VFA concentrations and higher resistance to Salmonella colonization than chicks reared on new litter.     

Changes in the Salmonella status of broiler chickens subjected to simulated shipping conditions.

Market-age broiler chickens from flocks infected with Salmonella typhimurium were killed after being placed in crates and subjected to simulated shipping conditions for five, 19 or 24 hours. The cloacal feces, ceca and exteriors of the birds were cultured for salmonellae to identify shedders, cecal carriers and external carriers respectively, and the results compared with those obtained from pen-mates killed at the time the tested birds were put into the crates. Carriage of S. typhimurium was significantly higher among birds placed in clean crates than among the uncrated controls, mainly due to an increase in cecal carriers (from 23.5% to 61.5%). This increase was not related to the time spent in the crate. Twenty-four chickens were placed in crates contaminated with Salmonella alachua and all 24 became carriers of this organism: 22 (91.5%) of these were cecal carriers and 18 (75%) were shedders. This contamination also spread to 15/24 chickens placed in clean crates and "shipped" in the same truck: six of these became cecal carriers and seven were shedders. These results indicated that chickens in shipping crates which are exposed to salmonellae under transport conditions may readily become infected and begin to shed salmonellae within 24 hours.     

Effect of reticuloendotheliosis virus on the response of chickens to Salmonella typhimurium infection

Groups of 25 chickens free of maternal antibody to reticuloendotheliosis virus (REV) were inoculated with either third or seventh passage REV at either one or seven days of age. Some of the birds inoculated at day 1 with REV were inoculated with Salmonella typhimurium either concurrently or six or 13 days later while some of those inoculated with REV at day 7 were inoculated concurrently with S typhimurium. At day old, infection with S typhimurium alone caused the death of 12 of 25 chicks whereas in the dual infection, using the third passage REV, 18 of 25 birds died. Similarly no seven or 14 day old chickens died when challenged with S typhimurium alone, but previous day-old infection with REV caused a respective mortality of eight of 25 and five of 25 birds. With the seventh passage REV a similar pattern was seen. At day old S typhimurium infection alone killed seven of 25 birds whereas combined with virus the mortality was 14 of 25 and while S typhimurium alone killed none of 25 chicks infected at seven days old, the mortality in birds also infected with REV was 14 of 25. Combined virus and bacterial infections did not increase the proportion of feathering defects in birds surviving S typhimurium infections. There was a significantly higher proportion of feathering defects in birds infected with third passage virus compared with seventh passage virus. Although a higher proportion of birds had antibody responses to REV in the seventh than in the third passage group, there was no discernible difference in the effect the different viruses had on chickens' susceptibility to S typhimurium.